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Optimizing reactions for qRT-PCR using regular thermocycler

qPCR optimization primer optimization template optimization

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#1 Neanderthal

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Posted 08 March 2013 - 04:32 AM

Hello,

I've been told that people optimize reactions for qPCR in a regular thermocycler. Is this comparable with the optimization done in an qRT-PCR machine (ex. ABI 7900HT fast real-time PCR machine)?

If this is possible, how do I go about optimizing my qPCR reactions in a normal PCR machine?

I guess by 'optimizing reactions of qPCR' means both template and primer optimization (with different cDNA template dilutions and different primer dilutions) and then running them in gel. Is this correct?

How do you interpret the results from gel images from such an experiment?

Thanks,

Kart

#2 wasabi

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Posted 08 March 2013 - 04:40 AM

Hi,

I think the most important is to optimize concentration of both primers and (if taqman) the probe. This has to be done on a real time cycler. On a regular cycler you can just see if there is nonspecific binding, so an extra band with unexpected size on a gel. Normally this won't be a problem when using software such as primer express.

greetings

#3 Trof

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Posted 08 March 2013 - 01:04 PM

The only thing that could be roughly estimated is annealing temperature, especially if you have a gradient cycler. But you need to use the same master mix as qPCR. but the primers should be designed to work with Ta 60 ideally, for compatibility if not other reasons. You would need to use cycle number commonly used for classic PCR, not qPCR, but at this point, qPCR is much more sensitive, so you may not catch the right cycle range, where the differences in efficiency are distinct.

I don't do it. Starting to optimize your reaction with SYBR can tell you precisely the ideal annealing, both regarding yield and speciity (checking meting curves all the time). Then if the reaction is probe based, you need to optimize primers and probe concentration, which can't be done on normal cycler as wasabi said.

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#4 Neanderthal

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Posted 11 March 2013 - 11:15 PM

Thanks alot Wasabi and Trof for your inputs. I'm using SYBR by the way. If I understood it right, regular PCR will only give if there is a nonspecific binding or not, and since the same master mix as qPCR should be used, there won't be any help with savingreagents also.

#5 Trof

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Posted 12 March 2013 - 01:17 AM

Technically you could run classic PCR on normal cycler with normal mix, to find the optimal annealing (yield) and nonspecifities, problem is the classic mix would behave different and SYBR is a PCR inhibitor which also affects the reaction. Mixes can also have different aditives inside, that modifies the reaction again.
So you would optimize, but that would be optimal only for the classic PCR and your mix and not real-time one, so actually useless and you would spent both classic and qPCR reagents.
If you had mixes from the same manufacturer, same enyzme and everything, that actually differ only in the presence of SYBR in the qPCR, it would be more meaningfull, but you need to optimize the final mix anyway.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon






Also tagged with one or more of these keywords: qPCR optimization, primer optimization, template optimization

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